Neurotransmitter-gated membrane ion channels are among the most important target sites of alcohol action in the nervous system, although the manner in which alcohols modulate the function of these transmembrane proteins has not been established. The aim of this project is to investigate the actions of alcohols and related compounds on neurotransmitter-gated ion channels thought to be involved in producing the intoxicating effects of alcohols in nervous tissue. The function of the N-methyl-D-aspartate (NMDA) receptor-channel, a type of receptor for the excitatory neurotransmitter glutamate, is inhibited by intoxicating concentrations of ethanol. Results of previous studies have established that alcohols inhibit NMDA receptors via an action on gating of the ion channel, largely by decreasing mean open time, rather than by influencing agonist or coagonist binding. Using electrophysiological patch-clamp recording, we have localized the probable site of ethanol action either to the extracellular region of the protein, or to a region of one of the transmembrane domains near the extracellular surface. We have also performed scanning mutagenesis in the membrane-associated domains of the NR1 and NR2 subunits of the NMDA receptor to identify regions that may influence ion channel gating, and thus could be sites of alcohol action. Although conservative mutations in the majority of amino acid positions in the membrane-associated domains produced little or no alteration in ion channel function or ethanol sensitivity, we have identified a position in one of the membrane-associated domains in which substitutions can produce marked changes in gating characteristics, including mean open time. Amino acid substitutions at this position also produce changes in ethanol sensitivity. These changes in ethanol sensitivity, however, do not appear to correlate strongly with ion channel gating parameters such as mean open time, nor do they appear to depend upon physicochemical parameters of the amino acid substituent such as molecular volume or hydrophobicity. The observed differences in alcohol sensitivity appear instead to be attributable to subtle structural differences in the amino acid substituent at the site. These results suggest that although ethanol and other alcohols inhibit NMDA receptors via changes in gating of the ion channel, manipulations that alter ion channel gating do not necessarily influence alcohol sensitivity of the NMDA receptor. Although a precise molecular site of alcohol action has yet to be found, these results are consistent with the existence of such a site, rather than with a nonspecific action of alcohol. Experiments in this unit have also been directed toward the structure and function of NMDA receptor membrane-associated domains, as well as the physiological regulation of inhibitory amino acid-gated ion channels.